Plant Cullin RING Ubiquitin E3 ligases (CRLs) play a critical role in targeted protein degradation, essential for physiological development and stress adaptation. The deneddylase activity of the COP9 signalosome (CSN) tightly regulates the cellular balance of neddylated cullins, which is crucial for maintaining the full spectrum of CRL functions. Although selective inositol polyphosphates (InsPs) act as cofactors in plant responses that involve ubiquitylation of negative regulators, their connection to CSN-CRL activities has remained unclear. In this study, we reveal that the two Arabidopsis thaliana InsP-kinases, IPK1 and ITPK1, physically interact and orchestrate the metabolic regulation of the CSN holo-complex activity. Notably, ITPK1 deficiency lowers Nedd8 processing rates, elevates the cellular ratios of neddylated cullins, and disturbs the dissociation equilibrium of CSN5 and CUL1 from the holo-complex. These findings uncover a novel autoregulatory switch in CSN functions, governed by deneddylation activity. Furthermore, we demonstrate that the phosphate starvation response (PSR), induced in phosphate-limited wild-type plants and constitutively active in the InsP-kinase mutants, is partly regulated by reduced deneddylation rates, which affect the stability of SPX4, a key negative regulator of PSR. Pharmacological inhibition of cullin neddylation stabilizes SPX4 and impairs PSR, thereby linking CSN-CRL dynamics to phosphate sensing. Conversely, pharmacologically inhibiting CSN5 deneddylase activity causes wild-type plants to exhibit PSR phenotypes similar to those of the InsP-kinase mutants. Collectively, these results reveal that specific InsP-kinases are partly involved in modulating plant PSR by fine-tuning the coordination between CRL and CSN activities.

The turnover of myo-inositol phosphates (InsPs) and myo-inositol pyrophosphates (PP-InsPs) is a dynamic process that plays an important role in many physiological processes by transmitting signals within cellular pathways and networks. Profiling the InsPs and PP-InsPs isomers and quantifying their change in abundance is a significant challenge for several reasons. First, InsPs and PP-InsPs constitute a diverse metabolite pool, characterized by the complexity as a result of the numerous possible isobaric isomers. Second, these species are usually of low abundance in biological samples. Third, they lack a chromophore, making UV or fluorescence detection unfeasible. Fourth, their high charge density and the instability of P-anhydride bonds make isolation and separation requirements particularly demanding. This chapter presents a capillary electrophoresis coupled to mass spectrometry (CE-MS) method as a powerful tool. It enables the separation of multiply charged InsPs and PP-InsPs with high resolution for profiling regioisomers with high sensitivity from biological samples.

E-cadherin downregulation is an epithelial-mesenchymal transition hallmark canonically attributed to transcriptional repression. Here we delineate a metabolite-driven endocytic route of E-cadherin downregulation in inflammation-associated colorectal cancer (CRC). Specifically, IP6 kinase-2 (IP6K2), a 5-diphosphoinositol pentakisphosphate (5-IP7) synthase upregulated in patients with CRC, is activated via a ROS-Src phosphorylation axis elicited by dextran sulfate sodium (DSS), generating 5-IP7 around adherens junction (AJ) to promote E-cadherin endocytosis and the transcriptional activities of β-catenin. Mechanistically, 5-IP7 inhibits inositol 5-phosphatases such as OCRL to promote PI(4,5)P2-mediated endocytic adaptor recruitment. Depleting 5-IP7 or overexpressing a 5-IP7 binding-deficient OCRL mutant confers resistance to DSS-elicited AJ disruption. Intestinal epithelium-specific IP6K2 deletion attenuates DSS-induced colitis/CRC, whereas an IP6K2 isoform-selective inhibitor protects wild-type but not IP6K2-/- mice against DSS insult. Thus, 5-IP7 is an oncometabolite whose stimulus-dependent synthesis relieves a PI(4,5)P2 dephosphorylation-based endocytic checkpoint, leading to AJ disassembly and protumorigenic β-catenin activation. Targeting IP6K2 could strengthen intestinal epithelial barrier against inflammation and cancer.

Phosphorylated myo-inositols (InsPs) are essential cytoplasmic signaling molecules, while their lipidated analogs (PtdInsPs) play a crucial role in membrane signaling. Stereoselective synthesis of these compounds has been achieved through various methods, predominantly using the meso compound myo-inositol as a starting material. However, phytate (InsP6), also a meso compound, is the most abundant inositol derivative in plants - far more prevalent than myo-inositol itself. Despite its abundance, phytate has been rarely used in synthetic strategies for accessing a variety of chiral inositol phosphates and their derivatives through selective dephosphorylations on a preparative scale. Here, we report gram-scale (stereo)selective dephosphorylations of phytate using phytases and demonstrate the application of these products in generating modified InsPs through a transient phosphitylation approach. Notably, the bacterial effector XopH efficiently desymmetrizes meso-phytate to yield enantiomerically pure 1-OH-InsP5. This transformation renders the 1-position accessible for further modifications, which, in biological systems, is where glycerolphosphate diesters are attached. By using selective dephosphorylations with phytases in concert with chemoselective telescoping reaction sequences, this approach greatly advances the stereoselective synthesis of inositol phosphates and their derivatives, such as glycerophosphoinositols, from abundant InsP6.

Inositol pyrophosphates (PP-InsPs) are highly phosphorylated signaling molecules that regulate diverse cellular processes, including phosphate homeostasis and energy metabolism across species. Despite extensive research on well-characterized exhaustively phosphorylated PP-InsPs, such as 5-PP-InsP5 (5-InsP7) and 1,5-(PP)2-InsP4 (1,5-InsP8), the functional relevance of less abundant not fully phosphorylated isomers, remains largely unknown. In this study, we synthesized all unsymmetric 5-PP-InsP4 isomers in enantiopure form and assigned their structures using ³¹P-NMR analysis in combination with a chiral solvating agent. Additionally, we developed ¹⁸O-labeled PP-InsP4 standards for quantitative mass spectrometry in combination with capillary electrophoresis (CE-MS), enabling the study of PP-InsP4 in Arabidopsis thaliana under phosphate starvation. Our findings show that the previously detected, phosphate starvation-induced root-specific PP-InsP4 isomer does not match any 5-PP-InsP4 isomer, contrary to previous suggestions, thus indicating an alternative phosphorylation pattern. Enzyme assays further demonstrate that Arabidopsis ITPK1 selectively phosphorylates [6-OH]-InsP5 and [3-OH]-InsP5 at the 5-position, while other InsP5 isomers remain unchanged. This suggests that an unidentified enzymatic activity is involved in the formation of the elusive root PP-InsP4 species. Our study provides a comprehensive framework for the synthesis, analysis, and functional investigation of PP-InsP4, providing an entry point for future studies on their biochemical activity and their physiological roles.

This study investigates the metabolic pathways of inositol pyrophosphates (IPPs) in the yeast cell line ΔSPX and the human tumor cell line HCT116. Utilizing pulse-labelling experiments with 18O water and ordinary differential equation (ODE) models, we explore the synthesis and turnover of the highly phosphorylated IPP, 1,5-InsP8. Our findings challenge the notion that 1,5-InsP8 can be synthesized through distinct routes, revealing a linear reaction sequence in both systems. Employing model reduction via the profile likelihood method, we achieved statistically concise identifiability analysis that led to significant biological insights. In yeast, we determined that 1,5-InsP8 production primarily occurs through the phosphorylation of 5-InsP7, with the pathway involving 1-InsP7 deemed unnecessary as its removal did not compromise model accuracy. In HCT116 cells, 1,5-InsP8 synthesis is mainly driven by 1-InsP7, with variations observed across different experimental conditions. These results underscore the utility of model reduction in enhancing our understanding of metabolic pathways, challenging traditional views of IPP metabolism, and providing a framework for future investigations into the regulation and implications of linear IPP pathways in eukaryotic cells.

Inositol pyrophosphates (PP-InsPs) are soluble cellular messengers that integrate environmental cues to induce adaptive responses in eukaryotes. In plants, the biological functions of various PP-InsP species are poorly understood, largely due to the absence of canonical enzymes found in other eukaryotes. The recent identification of a new PP-InsP isomer with yet unknown enantiomeric identity, 4/6-InsP7 in the eudicot Arabidopsis thaliana, further highlights the intricate PP-InsP signalling network employed by plants. Yet, the abundance of 4/6-InsP7 in land plants, the enzyme(s) responsible for its synthesis, and the physiological functions of this species are all currently unknown. In this study, we show that 4/6-InsP7 is ubiquitous in the studied land plants. Our findings demonstrate that the Arabidopsis inositol polyphosphate multikinase (IPMK) homologs, AtIPK2α and AtIPK2β phosphorylates InsP6 to generate 4/6-InsP7 as the predominant PP-InsP species in vitro. Consistent with this, AtIPK2α and AtIPK2β act redundantly to control 4/6-InsP7 production in planta. Notably, activity of these IPK2 proteins is critical for heat stress acclimation in Arabidopsis. Our parallel investigations using the liverwort Marchantia polymorpha suggest that the PP-InsP synthase activity of IPK2 and role of IPK2 in regulating the heat stress response are conserved in land plants. Furthermore, we show that the transcription activity of heat shock factor (HSF) is regulated by IPK2 proteins, providing a mechanistic framework of IPK2-controlled heat stress tolerance in land plants. Collectively, our study indicates that IPK2-type kinases have played a critical role in transducing environmental cues for biological processes during land plant evolution.

New antifungals are urgently needed to treat deadly fungal infections. Targeting the fungal inositol polyphosphate kinases IP3-4K (Arg1) and IP6K (Kcs1) is a promising strategy as it has been validated genetically to be crucial for fungal virulence but never pharmacologically. We now report the synthesis of DT-23, an analogue of N2-(m-trifluorobenzylamino)-N6-(p-nitrobenzylamino)purine (TNP), and demonstrate that it more potently inhibits recombinant Arg1 from the priority pathogen Cryptococcus neoformans (Cn) (IC50 = 0.6 μM) than previous analogues (IC50 = 10-30 μM). DT-23 also inhibits recombinant Kcs1 with similar potency (IC50 = 0.68 μM) and Arg1 and Kcs1 activity in vivo. Unlike previous analogues, DT-23 inhibits fungal growth (MIC50 = 15 μg/mL) and only 1.5 μg/mL synergizes with Amphotericin B to kill Cn in vitro. DT-23/Amphotericin B is also more protective against Cn infection in an insect model compared to each drug alone. Transcription profiling shows that DT-23 impacts early stages in IP synthesis and cellular functions impacted by IPK gene deletion, consistent with its targeted effect. This study establishes the first pharmacological link between inhibiting IPK activity and antifungal activity, providing tools for studying IPK function and a foundation to potentially develop a new class of antifungal drug.

Inositol pyrophosphates (PP-InsPs) are highly phosphorylated signaling molecules that regulate diverse cellular processes, including phosphate homeostasis and energy metabolism across species. Despite extensive research on well-characterized exhaustively phosphorylated PP-InsPs, such as 5-PP-InsP5 (5-InsP7) and 1,5-(PP)2-InsP4 (1,5-InsP8), the functional relevance of less abundant not fully phosphorylated isomers, remains largely unknown. In this study, we synthesized all unsymmetric 5-PP-InsP4 isomers in enantiopure form and assigned their structures using 31P-NMR analysis in combination with a chiral solvating agent. Additionally, we developed 18O-labeled PP-InsP4 standards for mass spectrometry in combination with capillary electrophoresis (CE-MS), enabling the assignment of PP-InsP4 in Arabidopsis thaliana under phosphate starvation. Our findings show that the previously detected, phosphate starvation-induced root-specific PP-InsP4 isomer does not match any 5-PP-InsP4 isomer, contrary to previous suggestions, thus indicating an alternative phosphorylation pattern. Enzyme assays further demonstrate that Arabidopsis ITPK1 selectively phosphorylates [6-OH]-InsP5 and [3-OH]-InsP5 at the 5-position, while other InsP5 isomers remain unchanged. This suggests that an unidentified enzymatic activity is involved in the formation of the elusive root PP-InsP4 species. Our study provides a comprehensive framework for the synthesis, analysis, and functional investigation of PP-InsP4, providing an entry point for future studies on their biochemical activity and their physiological roles.

Capillary electrophoresis mass spectrometry (CE-MS) allows for the rapid and accurate quantitative analysis of inositol phosphates (InsPs) and inositol pyrophosphates (PP-InsPs). The recent discovery of new InsPs and PP-InsPs isomers in plants and mammals necessitates new heavy isotope references for quantitative analysis of complex cellular extracts. Here, we evaluate 18O-labeled InsPs and PP-InsPs as alternatives to 13C labeled internal standards for quantitation by CE-MS. In contrast to 13C labels, the 18O labels are introduced at the end of a synthetic campaign and not at the beginning, rendering 18O much more accessible and affordable as a label. A series of 18O-labeled InsPs and PP-InsPs with different numbers and positions of 18O atoms were synthesized, enabling systematic investigation of MS2 fragmentation pathways. We propose two major dissociation pathways to elucidate the 18O redistribution of the dominant product ion (the loss of H3PO4). Based on these insights, we identified the loss of HPO3 as a suitable transition for minimizing isotope redistribution in MS2 analysis. The ratios of this alternative product ion and dominant product ion were reproducible across replicates, concentration, and measurement days, supporting the use of this alternative product ion as a reliable product ion for quantitative analysis. Application to Saccharomyces cerevisiae, HCT116 cells, and Arabidopsis thaliana extracts confirmed accurate quantitation and precision comparable to 13C-based methods.

Zusammenfassung Inositolpyrophosphate (PP‐InsPs) sind hochphosphorylierte Signalmoleküle, die in verschiedenen Organismen zentrale zelluläre Prozesse wie die Phosphathomöostase und den Energiestoffwechsel regulieren. Während gut charakterisierte, vollständig phosphorylierte PP‐InsPs wie 5‐PP‐InsP 5 (5‐InsP 7 ) und 1,5‐(PP) 2 ‐InsP 4 (1,5‐InsP 8 ) intensiv untersucht wurden, ist bislang kaum etwas über die Funktion wenig häufiger, nur partiell phosphorylierter Isomere bekannt. In dieser Studie wurden alle unsymmetrischen 5‐PP‐InsP 4 ‐Isomere in enantiomeren‐reiner Form synthetisiert und mithilfe von 31 P‐NMR‐Spektroskopie in Kombination mit einem chiralen Solvatisierungsreagenz strukturell zugewiesen. Zusätzlich entwickelten wir 18 O‐markierte PP‐InsP 4 ‐Standards für CE‐MS‐Analysen (Kapillarelektrophorese gekoppelt mit Massenspektrometrie), die die Identifizierung eines PP‐InsP 4 ‐Isomers in Arabidopsis thaliana unter Phosphatmangelbedingungen ermöglichten. Unsere Ergebnisse zeigen, dass das unter Phosphatmangel beobachtete, wurzelspezifische PP‐InsP 4 ‐Isomer entgegen früherer Annahmen keinem der synthetisierten 5‐PP‐InsP 4 ‐Isomere entspricht. Dies weist auf ein alternatives Phosphorylierungsmuster hin. Enzymatische Untersuchungen belegen zudem, dass Arabidopsis ITPK1 bevorzugt [6‐OH]‐InsP 5 und [3‐OH]‐InsP 5 an der 5‐Position phosphoryliert, während andere InsP 5 ‐Isomere unverändert bleiben. Dies legt nahe, dass eine bislang unbekannte enzymatische Aktivität an der Biosynthese des bislang nicht zugewiesenen PP‐InsP 4 ‐Isomers beteiligt ist. Unsere Studie bietet ein umfassendes methodisches Fundament für die Synthese, Analyse und funktionelle Charakterisierung von PP‐InsP 4 und eröffnet neue Perspektiven für die Erforschung ihrer biochemischen Eigenschaften und physiologischen Funktionen.

The homeostasis of intracellular inorganic phosphate is essential for eukaryotic metabolism and is regulated by the INPHORS signalling pathway, which employs inositol pyrophosphates (IPPs) as key intermediary messengers. This study investigates the metabolic pathways of inositol pyrophosphates (IPPs) in the yeast cell line PhoΔSPX and the human tumor cell line HCT116. Utilizing pulse-labelling experiments with 18O water and ordinary differential equation (ODE) models, we explore the synthesis and turnover of the highly phosphorylated IPP, 1,5-InsP8. Our findings challenge the notion that 1,5-InsP8 can be synthesized through distinct routes, revealing a linear reaction sequence in both systems. Employing model reduction via the profile likelihood method, we achieved statistically concise identifiability analysis that led to significant biological insights. In yeast, we determined that 1,5-InsP8 production primarily occurs through the phosphorylation of 5-InsP7, with the pathway involving 1-InsP7 deemed unnecessary as its removal did not compromise model accuracy. Crucially, this prediction of altered IPP concentrations was validated experimentally in vip1Δ and kcs1Δ knockout strains, providing orthogonal biological support for the reduced model. In HCT116 cells, 1,5-InsP8 synthesis is mainly driven by 1-InsP7, with variations observed across different experimental conditions. These results underscore the utility of model reduction in enhancing our understanding of metabolic pathways, coupling predictive modeling with experimental validation, and providing a framework for future investigations into the regulation and implications of linear IPP pathways in eukaryotic cells.

Inositol plays key roles in many cellular processes. Several studies focussed on the quantitative analysis of phosphorylated forms of inositol, enabled by analytical tools developed to detect these highly charged molecules. Direct measurement of free inositol however has been challenging, because the molecule is uncharged and polar. As a result, the mechanisms maintaining the homeostasis of the inositol remains poorly understood. In this study, we overcome these challenges by developing a quantitative liquid chromatography - mass spectrometry (LC-MS) protocol that can resolve and quantify the three main sugar molecules present inside cells: glucose, fructose, and inositol, as well as distinguish the clinically relevant isomers of inositol: myo-, scyllo-, and chiro-inositol. The quantitative power of the new method was validated by accurately monitoring the changes of inositol levels under well-established conditions in Saccharomyces cerevisiae, where the endogenous synthesis of inositol is increased in the transcription repressor OPI1 knockout opi1D and decreased when wild type yeast is fed with exogenous inositol. The method also revealed a new layer of regulation that takes place when exogenous inositol is added to further boost endogenous inositol synthesis in opi1D in a positive feedback loop. Analyses of mammalian cell lines provided many new insights into inositol metabolism. First, different cell lines displayed distinct sugar profiles and inositol concentrations and responded differently to inositol starvation. Second, mammalian cells can synthesize and import scyllo- but not chiro-inositol. Importantly, our method lent direct evidence to the previous hypothesis that lithium treatment could significantly reduce inositol levels in primary cortical neurons, thus diminishing the pool of free inositol available to the phosphoinositide cycle.

Land plants have evolved sophisticated sensing mechanisms and signaling pathways to adapt to phosphate-limited environments. While molecular players contributing to these adaptations in flowering plants have been described, how nonvascular bryophytes regulate phosphate (Pi) homeostasis remained largely unknown. In this study, we present findings that both male and female plants of the liverwort Marchantia polymorpha respond to altered phosphate availability through substantial developmental changes. We show that the second messenger inositol pyrophosphates (PP-InsPs) respond more quickly to changes in cellular Pi status than the lower inositol phosphates, highlighting a functional relationship between PP-InsP and Pi homeostasis in M. polymorpha. To further corroborate the possible involvement of PP-InsP in Pi homeostasis, we characterized M. polymorpha INOSITOL (1,3,4) TRIPHOSPHATE 5/6 KINASE1 (MpITPK1) that phosphorylates InsP6 to generate InsP7 both in vitro and in vivo. Consistent with the role of PP-InsPs in Pi homeostasis, M. polymorpha lines with enhanced MpITPK1 expression leading to the accumulation of 5-InsP7 and an InsP8 isomer, exhibit altered expression of phosphate starvation induced (PSI) genes and display attenuated responses to low phosphate. The characterization of MpPHO1-deficient plants with dramatically increased levels of 1,5-InsP8 further supports the role of PP-InsP in Pi homeostasis in this liverwort species. Notably, our study unveiled that MpITPK1 rescues the deregulated Pi homeostasis in Arabidopsis (Arabidopsis thaliana) ITPK1-deficient plants, suggesting that liverwort and eudicots share a functional ITPK1 homolog. In summary, our study provides insights into the regulation of Pi homeostasis by ITPK1-derived PP-InsPs in M. polymorpha. ITPK1-derived inositol pyrophosphates control phosphate homeostasis in the liverwort Marchantia polymorpha.

Plant yield is often maximized by the extensive use of mineral fertilizers, which, however, has severe environmental consequences. Phosphate is particularly problematic, as it represents a globally limited resource, and its runoff and soil erosion threaten open water bodies. Many crops engage in arbuscular mycorrhizal (AM) symbiosis with nutrient-acquiring fungi, aiding in the uptake of phosphate and other mineral nutrients. However, AM colonization is strongly reduced under high soil phosphate levels. A mechanistic understanding of phosphate sensing, phosphate starvation responses, and their connection to AM remains enigmatic. Here, we show that in Lotus japonicus , low-abundant, energy-rich inositol pyrophosphates act as master regulators of AM, orchestrating the crosstalk between phosphate starvation responses and plant root endosymbiosis. These findings hold promise for breeding nutrient-efficient crops. ### Competing Interest Statement The authors have declared no competing interest.

Dietary intake of phytate has various reported health benefits. Previous work showed that the gut microbiota can convert phytate to short-chain fatty acids (SCFAs), but the microbial species and metabolic pathway are unclear. Here we identified Mitsuokella jalaludinii as an efficient phytate degrader, which works synergistically with Anaerostipes rhamnosivorans to produce the SCFA propionate. Analysis of published human gut taxonomic profiles revealed that Mitsuokella spp., in particular M. jalaludinii, are prevalent in human gut microbiomes. NMR spectroscopy using 13C-isotope labelling, metabolomic and transcriptomic analyses identified a complete phytate degradation pathway in M. jalaludinii, including production of the intermediate Ins(2)P/myo-inositol. The major end product, 3-hydroxypropionate, was converted into propionate via a synergistic interaction with Anaerostipes rhamnosivorans both in vitro and in mice. Upon [13C6]phytate administration, various 13C-labelled components were detected in mouse caecum in contrast with the absence of [13C6] InsPs or [13C6]myo-inositol in plasma. Caco-2 cells incubated with co-culture supernatants exhibited improved intestinal barrier integrity. These results suggest that the microbiome plays a major role in the metabolism of this phytochemical and that its fermentation to propionate by M. jalaludinii and A. rhamnosivorans may contribute to phytate-driven health benefits. Mitsuokella jalaludinii and Anaerostipes rhamnosivorans degrade dietary phytate via synergistic interactions in the gut to produce the beneficial short-chain fatty acid propionate.

ABSTRACT Inositol tris/tetrakis phosphate kinases (IP 3-4 K) in the human fungal priority pathogens, Cryptococcus neoformans ( Cn Arg1) and Candida albicans ( Ca Ipk2), convey numerous virulence functions, yet it is not known whether the IP 3-4 K catalytic activity or a scaffolding role is responsible. We therefore generated a C. neoformans strain with a non-functional kinase, referred to as the dead-kinase (dk) Cn Arg1 strain (dkArg1). We verified that, although dk ARG1 cDNA cloned from this strain produced a protein with the expected molecular weight, dkArg1 was catalytically inactive with no IP 3-4 K activity. Using recombinant Cn Arg1 and Ca Ipk2 , we confirmed that, unlike the IP 3-4 K homologs in humans and Saccharomyces cerevisiae , Cn Arg1 and Ca Ipk2 do not phosphorylate the lipid-based substrate, phosphatidylinositol 4,5-bisphosphate, and therefore do not function as class I PI3Ks. Inositol polyphosphate profiling using capillary electrophoresis-electrospray ionization-mass spectrometry revealed that IP 3 conversion is blocked in the dkArg1 and ARG1 deletion ( Cnarg1 Δ) strains and that 1-IP 7 and a recently discovered isomer (4/6-IP 7 ) are made by wild-type C. neoformans . Importantly, the dkArg1 and Cnarg1 Δ strains had similar virulence defects, including suppressed growth at 37°C, melanization, capsule production, and phosphate starvation response, and were avirulent in an insect model, confirming that virulence is dependent on IP 3-4 K catalytic activity. Our data also implicate the dkArg1 scaffold in transcriptional regulation of arginine metabolism but via a different mechanism to S. cerevisiae since Cn Arg1 is dispensable for growth on different nitrogen sources. IP 3-4 K catalytic activity therefore plays a dominant role in fungal virulence, and IPK pathway function has diverged in fungal pathogens. IMPORTANCE The World Health Organization has emphasized the urgent need for global action in tackling the high morbidity and mortality rates stemming from invasive fungal infections, which are exacerbated by the limited variety and compromised effectiveness of available drug classes. Fungal IP 3-4 K is a promising target for new therapy, as it is critical for promoting virulence of the human fungal priority pathogens, Cryptococcus neoformans and Candida albicans , and impacts numerous functions, including cell wall integrity. This contrasts to current therapies, which only target a single function. IP 3-4 K enzymes exert their effect through their inositol polyphosphate products or via the protein scaffold. Here, we confirm that the IP 3-4 K catalytic activity of Cn Arg1 promotes all virulence traits in C. neoformans that are attenuated by ARG1 deletion , reinforcing our ongoing efforts to find inositol polyphosphate effector proteins and to create inhibitors targeting the IP 3-4 K catalytic site, as a new antifungal drug class.

Inositol pyrophosphates (PP-InsPs) are eukaryote-specific second messengers that regulate diverse cellular processes, including immunity, nutrient sensing, and hormone signaling pathways in plants. These energy-rich messengers exhibit high sensitivity to the cellular phosphate status, suggesting that the synthesis and degradation of PP-InsPs are tightly controlled within the cells. Notably, the molecular basis of PP-InsP hydrolysis in plants remains largely unexplored. In this study, we report the functional characterization of MpDDP1, a diadenosine and diphosphoinositol polyphosphate phosphohydrolase encoded by the genome of the liverwort, Marchantia polymorpha. We show that MpDDP1 functions as a PP-InsP phosphohydrolase in different heterologous organisms. Consistent with this finding, M. polymorpha plants defective in MpDDP1 exhibit elevated levels of 1/3-InsP7 and 1/3,5-InsP8, highlighting the contribution of MpDDP1 in regulating PP-InsP homeostasis in planta. Furthermore, our study reveals that MpDDP1 controls thallus development and vegetative reproduction in M. polymorpha. Collectively, this study provides insights into the regulation of specific PP-InsP messengers by DDP1-type phosphohydrolases in land plants.